Many pathogenic Gram-negative bacteria such as Escherichia coli, Haemophilus influenzae, Salmonella enteriditis, Salmonella typhimurium, Bordetella pertussis, Yersinia enterocolitica, Yersinia perstis, Helicobacter pylori and Klebsiella pneumoniae assemble hair-like adhesive organelles called pili on their surfaces. Pili are thought to mediate microbial attachment, often the essential first step in the development of disease, by binding to receptors present in host tissues and may also participate in bacterial—bacterial interactions important in biofilm formation.
Uropathogenic strains of E. coli express P and type 1 pili that bind to receptors present in uroepithelial cells. Adhesive P pili are virulence determinants associated with pyelonephritic strains of E. coli whereas type 1 appear to be more common in E. coli causing cystitis. The adhesin present at the tip of the pilus, PapG binds to the Gal (1–4)Gal moiety present in the glycolipids and glycoproteins, while the type 1 adhesin, FimH, binds D-mannose present in glycolipids and glycoproteins.
Type 1 pili are adhesive fibers expressed in E. coli as well as in most of the Enterobacteriaceae family. The type 1 pilus is a right handed helix with about 3 subunits per turn, a diameter of approximately 70 Å, a central pore of about 20–25 Å, and a rise per subunit of about 8 Å. See G. E. Soto et al., EMBO J, 17: 6155 (1998). Type 1 pili are composite structures in which a short tip fibrillar structure containing FimG and the FimH adhesin (and possibly the minor component FimF as well) are joined to a rod comprised predominantly of FimA subunits. See Jones et al., Proc. Natl. Acad. Sci. U.S.A., 92: 2081 (1995). The FimH adhesin mediates binding to mannose-oligosaccharides. See S. N. Abraham et al., Nature, 336: 682 (1988); K. A. Krogfelt et al., Infect. Immun., 58: 1995 (1990). In uropathogenic E. coli, this binding event has been shown to play a critical role in bladder colonization and disease.
Type 1 pilus biogenesis proceeds by way of a highly conserved chaperone/usher pathway that is involved in the assembly of over 25 adhesive organelles in the Gram-negative bacteria. See G. E. Soto and S. Hultgren, J. Bacteriol., 181: 1059 (1999). The usher forms an oligomeric channel in the outer membrane with a pore size of approximately 2.5 nm and mediates subunit translocation across the outer membrane. See D. G. Thanassi et al., Proc. Natl. Acad. U.S.A., 95: 3146 (1998).
P pili is a heteropolymeric surface fiber with an adhesive tip and consists of two major sub-assemblies, the pilus rod and the tip fibrillum. The pilus rod is a thick rigid rod made up of repeating PapA subunits arranged in a right-handed helical cylinder whereas the tip fibrillum is a thin, flexible tip fiber extending from the distal end of the pilus rod and is composed primarily of repeating PapE subunits arranged in an open helical configuration. Two components of the tip fibrillum, PapK and PapF, act as adaptors. PapK is thought to link the pilus rod to the base of the tip fibrillum and regulates the length of the tip fibrillum: its incorporation terminates its growth and nucleates the formation of the pilus rod. PapF is thought to join the PapG adhesin to the distal end of the flexible tip fibrillum.
The biogenesis of P pili also occurs via the highly conserved chaperone/usher pathway. See T. G. Thanassi et al., Curr. Opin. Microbiol., 1: 223 (1998); D. L. Hung et al., EMBO J, 15: 3792 (1996). P pili are adhesive organelles encoded by eleven genes in the pap (pilus associated with pyelonephritis) gene cluster found on the chromosome of uropathogenic strains of E. coli. Six genes encode structural pilus subunits, PapA, PapH, PapK, PapE, PapF and PapG. See S. J. Hultgren et al., Cell 73: 887 (1993).
In P pili, two of the genes in the pap operon, papD and papC, encode the chaperone and usher, respectively. Chaperones such as PapD in E. coli are required to bind to pilus proteins imported into the periplasmic space, partition them into assembly component complexes and prevent non-productive aggregation of the subunits in the periplasm. See Kuehn M. J. et al., Proc. Natl. Acad. Sci. USA 88: 10586 (1991). PapD is a periplasmic chaperone that mediates the assembly of P pili. Detailed structural analysis has revealed that the PapD chaperone is the prototype member of a conserved family of periplasmic chaperones in Gram-negative bacteria. Periplasmic chaperones consist of two immunogloblin-like domains with a deep cleft between the two domains. See A. Holmgren and C. I. Branden, Nature, 342: 248 (1989); M. Pellecchia et al., Nature Struct. Biol., 5: 885 (1998). Further, all members of the periplasmic chaperone superfamily have a conserved hydrophobic core that maintains the overall features of the two domains.
Periplasmic chaperones, along with outer membrane ushers, constitute a molecular mechanism necessary for guiding biogenesis of adhesive organelles in Gram-negative bacteria. These chaperones function to cap and partition interactive subunits imported into the periplasmic space into assembly competent co-complexes, making non-productive interactions unfavorable. The chaperone-subunit co-complexes are targeted to the outer membrane usher where subunits, or ushers, assemble in a specific order to form a pilus. During pilus biogenesis, PapD binds to and caps interactive surfaces on pilus subunits and prevents their premature aggregation in the periplasm. PapD binds to each of the pilus subunit types as they emerge from the cytoplasmic membrane and escorts them in assembly-competent, native-like conformations from the cytoplasmic membrane to outer membrane assembly sites comprised of PapC. PapC has been termed a molecular usher since it receives chaperone-subunit co-complexes and incorporates, or ushers, the subunits from the chaperone co-complex into the growing pilus in a defined order.
In the absence of an interaction with the chaperone, pilus subunits aggregate and are proteolytically degraded. Kolmer et al. and Jones et al. have shown that the DegP protease degrades pilus subunits in the absence of the chaperone. See J. Bacteriol. 178: 5925 (1996); BIBO, 16: 6394 (1997). This discovery led to the elucidation of the fate of pilus subunits expressed in the presence or absence of the chaperone using monospecific antisera in Western blots of cytosolic membrane, outer membrane and perplasmic proteins prepared according to methods known in the art.
Thus, prevention or inhibition of normal pilus assembly in Gram-negative bacterium impacts the pathogenicity of the bacterium by preventing the bacterium from attaching to and infecting host tissues. Moreover, changes in the binding between pilus subunits and chaperones can have a dramatic impact on the efficiency of pilus assembly, and thus on the ability of Gram-negative bacterium to adhere to and consequentially, infect host tissues. Prevention and inhibition of binding between pilus subunits and between pilus subunits and periplasmic chaperones have the effect of impairing pilus assembly, whereby the infectivity of the Gram-negative bacterium expressing the pili is reduced. Accordingly, a need exists, in general, for compositions and methods for preventing or inhibiting the normal interaction between pilus subunits and/or between a pilus subunit and a chaperone.
However, identification of such compositions has heretofore relied on serendipity and/or systematic screening of large numbers of natural and synthetic compounds. A far superior method of drug-screening relies on structure-based drug design. The three dimensional structures of proteins or protein fragments are determined and potential agonists and/or potential antagonists are designed with the aid of computer modeling. However, heretofore the three-dimensional structure illustrating the interaction between pilus subunits and/or between a pilus subunit and a chaperone has remained unknown, essentially because no such protein co-crystals had been produced which would permit the required X-ray crystallographic data to be obtained.
Therefore, there is presently a need for obtaining a co-crystal of a co-complex of a pilus and a chaperone to allow such crystallographic data to be obtained. Furthermore there is a need for the determination of the three-dimensional structure of such co-crystals. Finally, there is a need for procedures for related structural based drug design based on such crystallographic data.